There is rapidly increasing interest in developing molecular imaging approaches that enable traditional radiological imaging techniques to obtain a wide range of information about molecular and cellular processes that occur in normal and diseased tissue. A range of information is considered important such as the ability to monitor cell migration, the development of reporters that enable imaging of gene expression, the development of robust strategies to image receptors, and the development of environmentally sensitive agents that can be used to detect the presence of specific enzymes or monitor changes in ion status such as increases in intracellular calcium. The long term goals of this work are to develop strategies that enable MRI contrast that is sensitive to a wide range of molecular and cellular processes. This work builds on over 15 years of work where we have demonstrated the first MRI strategy for detecting gene expression, the first MRI approach for monitoring a surrogate of calcium influx, the first MRI approach for performing neuronal track tracing, and the first MRI approach for monitoring the migration of single cells in vivo. These all represented initial reports by any radiological imaging technique which enabled these processes to be measured. These techniques are finding widespread application to imaging pre-clinical models of a broad range of diseases. Based on this past track record of success we continue to look for novel ways to develop MRI contrast specific for biological processes. Over the past year we have made progress in all of the specific aims. [unreadable] [unreadable] Aim 1: Develop iron oxide based contrast for labeling and imaging the migration of endogenous neural stem cells. Over the past few years we have demonstrated the unique advantages of micron sized iron oxide particles for MRI of specific cells. Single cells can be detected and indeed, single particles within single cells can be detected. The main paradigm for MRI of cell migration is to label cells ex vivo and monitor migration after transplantation into an animal. These studies have traditionally required very efficient labeling using nano sized particles. The ability to detect a single particle enables inefficient labeling strategies. In particular we have demonstrated that injection of particles into the ventricles of the rat brain enables particles to be taken up by neural precursors in the subventricular zone and MRI can monitor the migration of cells to the oflactory bulb. Over the past year we have demonstrated that the micron particles can be used as carriers of fluorescent molecules that enable cells to be labeled for morphological studies or with calcium sensitive dyes to enable monitoring of calcium transients. Furthemore, we have quantitated the distribution of cells that are labeled and have found all expected cell types in the rostral migratory stream labeled to about 30% efficiency. Inhibition of neuronal precursor production demonstrated that contrast detected in MRI was due to migration of precursor cells. In the coming year we will develop techniques for more efficiently delivering particles to precursor cells. We will determine if single cells can be detected and we will develop techniques that enable quantitation of the distribution of new cells in the olfactory bulb. Furthermore we will test if this labeling strategy enables monitoring cell migration to sites of disease (tumors), damage (stroke), or areas where plasticity is occuring. [unreadable] [unreadable] Aim 2: Apply microfabrication techniques to manufacture unique metal structures that may be valuable for MRI contrast. Iron oxide particles commonly used for MRI are very potent contrast agents enabling detection of single mciron sized particles. However, due to bulk phase manufacture of particles they are not very uniform and they do not contain very high content of metal. A solution to this problem is to use modern microfabrication techniques to manufacture metal based, micron sized contrast agents. To begin this work we have explored a variety of approachs to microfabrication of MRI contrast agents. We have demonstrated that precise definition of shapes and spacing of microfabricated structures leads to novel MRI agents. As expected mciron sized microfabricated nickel structures are very potent MRI contrast agents. Microfabrication gives us a great deal of flexibility to make structures that may have novel uses. For example, particles spaced at distances much smaller than an MRI voxel can be distinguished and water associated with properly designed structures can be distinguished. These initial results are all in phantoms and over the coming year we will develop strategies that enable us to label cells with these microfabricated particles. [unreadable] [unreadable] Aim 3: Develop novel delivery mechanisms to extend the applicability of manganese enhanced MRI. Over the past ten years we have demonstrated the remarkable utility of the manganese ion for MRI contrast. Manganese ion enters cells on ligand or voltage gated calcium channels and so can be used as an MRI agent to monitor calcium influx. Once inside of neurons, manganese will move in an anterograde direction and cross functional synapses enabling neuronal networks to be imaged with MRI. Finally, manganese given systemically gives cytoarchitectural information about the rodent brain. These successed have us interested in broadening the ways in which manganese ion can be delivered to cells. Over the past year we have made transferrin-manganese complexes. When bound to transferrin manganese is a poor MRI contrast agent. However, when transferrin is taken up by cells it can release manganese which is then trapped intracellularly. Thus, transferrin manganese is an agent that monitors the successful endocytosis of the transferrin by its receptor. Experiments in hepatocytes and in brain demonstrate that this strategy is succesful and gives efficient contrast. We have begun to hunt for small molecular weight chelates that mimic transferrin in that they are poor MRI contrast agents until endocytosis occurs after which they would activate. This would enable coupling to any molecule that can recognize a target and trigger endocytosis. It is hypothesized that the combined targetting plus effective endoctosis will add a level of specificity to targeted contrast agents. In addition, the fact that manganese has a final intracellular distribution increases its efficacy as a contrast agent. Furthermore, we will begin to engineer the transferrin binding site so that it can be readily fused to other domains to recognize other receptors. [unreadable] [unreadable] Aim 4: Develop strategies that enable cellular processes to alter the relaxivity of MRI contrast agents. In specific aim 3 we demonstrated a way in which a normal biological process (endocytosis of transferrin-Mn) can alter the effectiveness of an MRI contrast agent. It would be very exciting to find ways in which this can occur which are sensitive to other biological processes. To this end we have simulated the effects of changing spacing of ferritin, a known biologically occuring iron oxide particle. These simulations show that the effects of ferritin on MRI signal are very sensitive to the specific spacing of ferritin molecules. This opens the possibility of coupling ferritin to molecules that change aggregation state to make MRI reporters of cellular processes. In particular, we have demonstrated in vitro that ferritn-actin fusions can make MRI sensitive to the state of actin polymerization. It is well known that the state of the cytoskeleton reports on a wide range of biological states. The results with ferritin open the possibility of having an MRI reporter of cytoskeleton or of cytoskeleton binding proteins.